Co-evolution of atypical BRAF and KRAS mutations in colorectal tumorigenesis.

BRAF mutations in colorectal cancer (CRC) comprise three functional classes: Class 1 (V600E) with strong constitutive activation, Class 2 with pathogenic kinase activity lower than Class 1, and Class 3 which paradoxically lacks kinase activity. Non-Class 1 mutations associate with better prognosis, microsatellite stability, distal tumour location and better anti-EGFR response. Analysis of 13 CRC cohorts (n=6,605 tumours) compared Class 1 (n=709, 10.7% of CRCs), Class 2 (n=31, 0.47%) and Class 3 (n=81, 1.22%) mutations. Class 2- and Class 3-mutant CRCs frequently co-occurred with additional Ras pathway mutations (29.0% and 45.7% respectively vs 2.40% in Class 1, p<0.001), often at atypical sites (KRAS non-codon 12/13/61, NRAS, or NF1). Ras pathway activation was highest in Class 1 and lowest in Class 3, with greater distal expression of EGFR ligands (AREG/EREG) supporting weaker BRAF driver mutations. Unlike Class 1 mutants, Class 3 tumours resembled chromosomally-unstable CRCs in mutation burdens, signatures, driver mutations and transcriptional subtypes, while Class 2 mutants displayed intermediate characteristics. Atypical BRAF mutations were associated with longer overall survival than Class 1 (HR=0.25, p=0.011), but lost this advantage in cancers with additional Ras mutation (HR=0.93, p=0.86). This study supports the suggestion that Class 3 BRAF mutations amplify existing Ras signalling in a two-mutation model and that enhancement of weak/atypical Ras mutations may suffice for tumorigenesis, with potentially clinically-important heterogeneity in the Class 2/3 sub-group. Implications: The heterogeneous nature of BRAF-mutant CRCs, particularly among Class 2/3 mutations which frequently harbour additional Ras mutations, highlights the necessity of comprehensive molecular profiling.